HLA-B*27 typing by group-specific hybridization in microtiter plates

Abstract: We have developed and evaluated a test for HLA-B*27 based on PCR and DNA hybridization in microtiter plates. A region within exon 2 of the HLA-B gene is amplified and labeled by PCR and the amplification product is hybridized to a group-specific HLA-B*27 and a generic control oligonucleotide probe in two separate cavities of the plate. Bound sequences are detected using an ELISA-like protocol. The assay has been evaluated on 254 DNA samples routinely received for B27 testing in parallel with sero-logical and SSP-PCR typing. Results were concordant in typing 102 HLA-B27-positive and 152 HLA-B27-negative individuals except for two samples containing HLA-B*73, which stained B27 positive in the microwell test. The new procedure is rapid and simple to perform, and the microwell format is particularly suitable for automation.     

Rapid detection of all HLA-B*27 alleles (B*2701-B*2725) by group-specific polymerase chain reaction

Human leucocyte antigen-B*27 is strongly associated with a number of rheumatic diseases, including ankylosing spondylitis and reactive arthritis. Targeted detection of the B*27 group by molecular methods is hampered by the extreme heterogeneity of the serological B*27 group. Here, we describe a simple, rapid sequence-specific primer-based method for detection of all 28 B*27 alleles defined to date. The method involves an initial screening with two sequence-specific polymerase chain reactions (PCRs), which has to be followed by two additional PCR amplifications in samples carrying a few rare subtypes of B*27, B*4202 or B*7301. The described protocol should be useful for laboratories involved in diagnostics and research of rheumatoid diseases.     

HLA-B40, B18, B27, and B37 allele discrimination using group-specific amplification and SSCP method

We developed a system for discriminating HLA-B40, B18, B27, and B37 alleles using a two-step PCR method followed by SSCP analysis. Fragments (0.8 kb) including exon 2, intron 2, and exon 3 were amplified in the first PCR. We used two sets of primers, one specific for HLA-B60-related alleles and the other specific for HLA-B6l-related, B18, B27, and B37 alleles. No amplifications of other class I genes or pseudogenes were observed. In the second PCR, exon 2 and exon 3 were amplified separately, using diluents of the first PCR products as templates. HLA-B6l-related, B18, B27, B37, and B60-related alleles were clearly discriminated in the SSCP analysis of the second PCR products. In a population study in which B6l alleles were analyzed, B^*4003 was detected in two Japanese individuals in addition to two B6l alleles previously reported to occur in Japanese, B^*4002 and B^*4006. The relative frequencies of B^*4002, B^*4006, and B^*4003 in Japanese were 58, 35, and 6%, respectively. The individuals having B^*4003 are the first non-South Americans in whom this allele has been detected. The SSCP banding patterns of 18 HLA-B60-positive Japanese population samples were identical to those of a B^*40012 sample for both exon 2 and exon 3. We also demonstrated that the B37 allele occurring in some Japanese is B^*3701.     

HLA-B*27 typing by sequence specific amplification without DNA extraction.

HLA-B27 appears to play a direct role in the pathogenesis of ankylosing spondylitis and almost all patients with this disease have HLA-B27. Therefore, a diagnosis of ankylosing spondylitis can virtually be excluded in the absence of HLA-B27. Many techniques have been used for HLA-B*27 typing. Of these, molecular methods are the most sensitive and specific but require extracted DNA as the testing material. A technique where HLA-B*27 is amplified directly from whole blood using sequence specific primers has been developed. This technique uses small sample volumes, is not restricted by choice of anticoagulant or sample age up to at least six weeks, and can be applied to other clinical polymerase chain reaction based procedures.     

A comprehensive approach for typing the alleles of the HLA-B locus by automated sequencing

Abstract: We describe an approach for typing alleles of the HLA-B locus by using automated sequencing technology. The exon 2 and exon 3 nucleotide sequence of each allele is determined directly from genomic DNA in two steps. In the first step, HLA-B exon 2, intron 2 and exon 3 sequences are amplified with one or two primer pairs out of a panel of 5 primer pairs that describe all known HLA-B alleles. In the second step, templates are sequenced in 5' and 3' orientations in a PCR assay that utilizes Taq polymerase to incorporate fluorescent dye-labeled nucleotides into each new strand synthesized. Gel electrophoresis of the labeled products is performed in an automated DNA sequencer. The derived sequences are aligned against reference sequences and each nucleotide position is evaluated for homology to consensus sequence. Using this strategy, the HLA-B allele sequence is directly ascertained with precision and efficiency. The automated sequencing strategy can be readily applied in the clinical laboratory as a practical tool for high resolution typing of HLA-B alleles.     

HLA-B*27 typing by sequence specific amplification without DNA extraction.

HLA-B27 appears to play a direct role in the pathogenesis of ankylosing spondylitis and almost all patients with this disease have HLA-B27. Therefore, a diagnosis of ankylosing spondylitis can virtually be excluded in the absence of HLA-B27. Many techniques have been used for HLA-B*27 typing. Of these, molecular methods are the most sensitive and specific but require extracted DNA as the testing material. A technique where HLA-B*27 is amplified directly from whole blood using sequence specific primers has been developed. This technique uses small sample volumes, is not restricted by choice of anticoagulant or sample age up to at least six weeks, and can be applied to other clinical polymerase chain reaction based procedures.     

Different linkage disequilibria of HLA-B27 subtypes and HLA-C locus alleles

Subtypes of HLA-B27 have been identified by cellular, serological and biochemical techniques. Comparison of the various B27 subtype designations showed the existence of seven B27 subtypes. The new WHO nomenclature (1987) of the B27 subtypes is included. We further report on different linkage disequilibria (ld) of the B27 subtypes. In Caucasoids, the prevalent subtype B27.5 is in ld with Cw1 and Cw2, whereas B27.2 is linked only with Cw2. In Orientals, the most frequent subtypes B27.4 and B27.6 usually occur with Cw3 or Cw blank; B27.5 mostly occurs with Cw2, and B27.2 is almost absent.     

Analysis of HLA-B*44 alleles encoded on extended HLA haplotypes by direct automated sequencing

Abstract: We developed a PCR-based approach to sequence exons 2 and 3 of HLA-B44 alleles from genomic DNA. We applied this method to determine the B44 alleles encoded on extended HLA-A, B, DRB1, DQB1 haplotypes and the degree of mismatching for B44 alleles among marrow transplant patients and their unrelated donors (URD). A total of 81 samples was studied and included 38 patients, 42 donors and the cell "FMB"; the 80 clinical samples were comprised of 8 unpaired patients, 12 unpaired donors, and 30 URD-recipient pairs. Three alleles encoding B44 were identified, B*4402 (N=51), 4403 (N=32) and a new allele designated B*44KB and named B*4405 (N=4). Of the 27 patients for whom family study was available, there were 13 different B*4402, 7 different B* 4403 and 2 new B*4405 haplotypes. HLA-A2, Cw*0501, B*4402, DRB1* 0401, DQB1*0301 (n=2); A2, Cw*0501, B*4402, DRB1*1501, DRB5* 0101, DQB1*0602 (n=2); and HLA-A29, Cw*1601, B*4403, DRB1* 0701, DQB1*0201 (n=5) comprised the most common patient haplotypes. Of 30 URD-recipient transplant pairs studied, 27 were HLA-A, B serologically matched and DRB1, DRB3, DRB5, DQB1 allele matched, and 3 pairs were DRB1-mismatched. All B44 allele mismatching (N=3) occurred among the 27 matched pairs. The novel B*4402-variant sequence, HLA-B*4405, was identified in 4 individuals, and in each case was associated with an HLA-B44, Cw*02022, DRB1*0101, DQB1*0501 haplotype. HLA-B*4405 and B*4402 are identical in exon 2; in exon 3 however, B*4405 encodes T instead of G at nucleotide position 75 which translates to a substitution of tyrosine for aspartic acid at codon 116. Finally, the published B*4402 sequence derived from cell "FMB" was found to contain an error; the corrected B*4402 sequence encodes G rather than C at position 146 of exon 3.     

Haplotype-specific amplification and nucleotide sequencing reveal the novel allele HLA-B*080104

The novel allele human leukocyte antigen (HLA)-B*080104 differs from HLA-B*080101 by a synonymous nucleotide exchange at position 231 in exon 2 (C-->T).     

Molecular analysis of HLA-B35 alleles and their relationship to HLA-B15 alleles

The HLA-B35 serotype is one of the largest allelic groups of HLA class I molecules and includes four isotypes. Of the four, the B35 variant isoform is relatively rare and is the most acidic form. DNA sequencing of die rare isoforms revealed three alleles, B*1522, B*3511, and B*3517. A phylogenetic tree of HLA-B15- and HLA-B35-related alleles for the exon 2 and 3 nucleotide sequences showed that exon 2 of B* 1522 clusters with B35 alleles whereas exon 3 clusters with B15 alleles. Branches of the tree suggest that the serodeterminants of B35, B62, B63, and B70 may reside in the αl domain, encoded by exon 2. The B*1520 and B*1522 genes, which type as B62 and B35, respectively, are hybrid molecules alternatively using exon 2 and exon 3 sequences of B*3501 and B*1501. A comparison of intron 2 sequences for B*3501, B*1501 and B*1522 suggests that the recombination site may have been in the region at the 3' end of intron 2. Despite being flanked by two highly polymorphic exons (exons 2 and 3), intron 2 is relatively well conserved in the B-locus, and it is characterized by seven to eight tandem repeats of the CGGGG pentanucleotide. A high degree of sequence homology and repetitive sequences are essential for a significant frequency of recombination. In this report, we reveal more about the complex evolutionary history of the HLA-B alleles     

Characterization of two new HLA-B alleles by sequence-based typing: HLA-B*0817 and HLA-B*1311

In this brief communication we report the characterization of two new HLA-B variants officially named HLA-B*0817 and HLA-B*1311. The HLA-B*0817 allele was identified in a Caucasoid male candidate for renal transplantation in the North Italy Transplant program. The nucleotidic sequence of exons 2, 3 and 4 of this novel allele is identical to that of HLA-B*0804 except for three point mutations in exon 2: from A to G at position 259, from C to G at position 261 and from G to A at position 302. These mutations are responsible for two aminoacidic substitutions [Asn (r) Glu, codon 63, and Ser (r) Asn, codon 77]. HLA-B*1311 was found in a volunteer donor belonging to National Marrow Donor Program(R). This new variant is identical to that of HLA-B*1301 except for three nucleotide substitutions at positions 353, 355 and 369 leading to two aminoacidic variations from Ile to Thr at codon 94 and from Ile to Leu at codon 95 and a silent mutation at codon 99.     

Typing for all known MICA alleles by group-specific PCR and SSOP

Major histocompatibility complex class I chain-related gene (MICA) is a recently discovered polymorphic gene in the HLA region expressed mainly by certain epithelial cells, keratinocytes, endothelial cells, fibroblasts, and monocytes. MICA is structurally quite different from the HLA class I genes and is potentially associated with some diseases and with immune response to transplants. Some DNA-based typing techniques have previously been described for MICA including sequence-based typing (SBT) and analysis of single strand conformational polymorphisms (SSCP). In the present experiments we have developed a strategy that allows identification of all 54 MICA alleles described so far, using group-specific polymerase chain reactions (PCR) and sequence-specific oligonucleotide probes (SSOP). To analyze for the polymorphisms in exons 2, 3, and 4 an initial screening with group-specific primers, based on polymorphism at position 69 of exon 2, and at position 615-616 of exon 4, was used to determine four major groups of alleles. Then group-specific PCR amplifications were performed and the amplified DNA was hybridized with the corresponding panels of SSOP. An additional amplification was performed with locus-specific primers and hybridized with a set of SSOP to identify and/or confirm the presence of some of the alleles. Unequivocal MICA typing was achieved for 97 of 103 individuals. Of 54 previously described alleles, only 14 were observed in this population. One unexpected hybridization pattern was observed, and molecular cloning and sequencing confirmed it to be a novel sequence, which was given the local designation MICA-055D. The gene frequencies among 103 unrelated North American Caucasian donors were determined and the linkage disequilibrium between MICA and HLA-B was analyzed.     

HLA-B27等位基因和亚型与强直性脊柱炎关系研究进展

大量的研究证明,强直性脊柱炎(AS)是与人类白细胞抗原(HLA)相关性最强的疾病。AS的发病与HLA-B27阳性密切相关,并与B7、B13、B40等几个等位基因有一定关系。HLA-B位点有42个等位基因,其中HLA-B27具有高度多态性,含有22个以上的亚型,不同亚型的碱基序列间只有个别差异。B27亚型在AS患者中的分布因地区和种族上的差别而不同,在中国主要以B2704和B2705为主,但以B2705分布最广。这几年大量的人B27转基因鼠实验证明AS与B27的关联性。     

Complete subtyping of the HLA-A locus by sequence-specific amplification followed by direct sequencing or single-strand conformation polymorphism analysis

Abstract: A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR-based HLA-A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA-A locus. The method is based on a sequence-specific amplification identifying the serologically defined HLA-A specificities. The PCR products generated by these group-specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single-strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities A1-A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of A1-A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid-phase sequencing or SSCP analysis of the group-specific PCR fragments allowed complete subtyping of the HLA-A locus. This strategy can identify all 48 HLA-A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA-A locus. Only 4 out of these 1128 allele combinations remained unresolved.     

Identification of two new HLA-B alleles, HLA-B*0732 and HLA-B*5809, by sequence-based typing

Here, we have described the characterization of two novel human leukocyte antigen-B (HLA-B) alleles. The new alleles, HLA-B*0732 and HLA-B*5809, were identified in Italian Caucasian individuals. B*0732 differs from HLA-B*0708 by one nucleotidic change at position 412 (from G to A) in exon 3, leading to an amino acidic substitution from Asp (GAC) to Asn (AAC) at codon 114. The sequence of B*5809 is identical to that of HLA-B*5801, except for a point mutation at position 583 in exon 3, where a T is substituted by a C. This change leads to an amino acidic substitution from Tyr (TAC) to His (CAC) at codon 171.     

Analysis of the complete cDNA sequences of HLA-DRB1 alleles with group-specific amplification primers in the Chinese Han population

Currently for the majority of HLA-DRB1 alleles the focus has been mainly on exon 2 and complete cDNA sequences of HLA-DRB1 alleles are rare. In this study, we analyzed the complete coding sequences of partial alleles of HLA-DRB1 locus. The cDNA was amplified by polymerase chain reaction using the group-specific primers located in the 5'- and 3'-untranslated regions to obtain the complete coding sequences. The amplification products were sequenced using an ABI BigDye? Terminator Cycle Sequencing kit. The HLA-DRB1 allele phylogenetic tree was analyzed by dnaman software. Full-length cDNA sequences of 22 HLA-DRB1 alleles were obtained in this study. HLA-DRB1*08:09, DRB1 *12:02:01, and DRB1*13:12 alleles were first reported for complete coding sequences. The sequences of exon 1 of HLA-DRB1*04:06:01, DRB1*08:03:02, and DRB1 *14:07:01 were newly presented. The complete coding sequences of HLA-DRB1 *01:01:01, DRB1*03:01:01:01, DRB1*04:01:01, DRB1*04:05:01, DRB1*07:01:01: 01, DRB1*09:01:02, DRB1*10:01:01, DRB1*11:01:01, DRB1*12:01:01, DRB1*13: 01:01, DRB1*13:02:01, DRB1*14:04, DRB1*14:54, DRB1*15:01:01:01, DRB1*15: 02:01, and DRB1*16:02:01 were identical to those previously reported. Forty polymorphic positions in complete coding sequences outside exon 2 of these HLA-DRB1 alleles were confirmed. According to the phylogenetic tree of full-length coding sequence, the HLA-DRB1 allele was classified into seven major allelic lineages. In conclusion, a protocol for HLA-DRB1 cDNA amplification and sequencing was improved and the data may help to determine the polymorphism of coding sequences outside exon 2.     

Differential enhancement of HLA-B27 by interferon.

To further examine the role of the HLA-B27 antigen in the pathogenesis of disorders such as anterior uveitis and ankylosing spondylitis, we have measured the level of enhancement of this antigen on peripheral blood lymphocytes and compared it with that of the class I HLA antigens HLA-A2 and HLA-B7 following interferon treatment. We found that the level of enhancement of HLA-B7 was greater than that of HLA-A2 or -B7 following treatment by alpha or gamma interferon (p less than 0.002). Similarly, the level of enhancement of HLA-B7 was greater than that of HLA-A2 (p less than 0.002) (Mann Whitney U-test). A differential enhancement of HLA-B27 by lymphokines may be important in the pathogenesis of HLA-B27-related disorders.